Protective anti-tumor immunity is impaired by immunosuppressive mechanisms. Immune checkpoint proteins, including CTLA-4, PD-1, and B7-H4, function as effector molecules to disable T-cell responses against cancer. Although checkpoint blockade using monoclonal antibodies (mab) have shown positive outcomes in clinical trials, the overall response rate has been disappointingly as low as 6-21%. Therefore, identifying novel checkpoint proteins is critically needed. We have discovered and functionally characterized a new Ig-superfamily inhibitory ligand, designated V-domain Immunoglobulin Suppressor of T cell Activation (VISTA). We hypothesize that VISTA functions as an additional and crucial immune-checkpoint ligand that controls anti-tumor immunity. The goal in this grant proposal is to determine the molecular and cellular mechanisms of VISTA-mediated immune suppression, both via its direct effect on T effector cell activation, and via its regulatory functon on other immunosuppressive cell types, which in turn control T effector cell responses against cancer. Accordingly, the specific Aims are: (1) Determine the molecular mechanisms whereby VISTA suppresses T-cell activation, and how it collaborates with another immune-checkpoint pathway PD-L1/PD-1 to suppress tumor-specific T-cell responses. (2) Determine the role of VISTA on the development and function of Foxp3+CD4+ Tregs. (3) Define the role of VISTA on the development, differentiation, and function of mononuclear phagocytes in the normal physiological state and during tumorigenesis. A collection of novel reagents and models including VISTA mab, VISTAKO mice, and VISTA conditional KO mice will be used for this study. In addition to a transplantable melanoma B16F10 model, we will employ a clinically relevant inducible-melanoma model for mechanistic studies and assessing VISTA-based therapeutic strategies. Impact: Any successful cancer immunotherapeutic strategy must consider the negative immune regulators that prevent the development of optimal anti-tumor immunity. As a novel immune checkpoint pathway, VISTA provides a new target for the immune intervention in cancer. This study will provide answers regarding VISTA- mediated immune regulation during tumorigenesis. It will establish a novel paradigm in which VISTA and PD- L1/PD1 synergize to control T-cell responses, thus providing a rationale for targeting VISTA either alone or in combination with other immune checkpoint pathways for cancer immunotherapy. Further, this study will establish a new paradigm regarding how tumors utilize VISTA to alter the differentiation and functions of Tregs and monocytes, thus providing novel strategies for targeting these prominent immune-suppressors in cancer immunotherapy.